“Isoprenoids” is a generic name for a variety of compounds, including sterols, carotinoids and terpenes, among others. Among them, there is a group of prenyl diphosphate compounds containing a coenzyme Q side chain, and the synthesis thereof depends on the polymerization-like condensation reaction of isopentenyl diphosphate, which is an isoprene unit containing 5 carbon atoms, as catalyzed by a prenyl diphosphate synthase.
The respective prenyl diphosphate synthases are roughly classified into 4 groups.
The short chain (3 or 4 isoprene units) prenyl diphosphate synthases are known to perform their catalytic function in the form of homodimers. Examples of such are farnesyl diphosphate synthase (Eberthardt, N. L. (1975), J. Biol. Chem., 250, 863-866) and geranylgeranyl diphosphate synthase (Sagami, H. (1994), J. Biol. Chem., 269, 20561-20566).
The medium chain (6 or 7 isoprene units) prenyl diphosphate syntheses are known to be heterodimeric enzymes composed of two proteins each independently having no catalytic activity. Examples are hexaprenyl diphosphate synthase (Fujii, H. (1982), J. Biol. Chem., 257, 14610), and heptaprenyl diphosphate synthase (Takahashi, I. (1980), J. Biol. Chem., 255, 4539).
Further, as for the long-chain (8 to 10 isoprene units) prenyl diphosphate syntheses, it is reported that prokaryote-derived such enzymes are undissociable homodimers and are activated by a polyprenyl diphosphate carrier protein (Ohnuma, S. (1991), J. Biol. Chem., 266, 23706-23713). At present, however, there is no report available about eukaryotes-derived long-chain prenyl diphosphate syntheses.
Coenzymes Q are composed of a quinone skeleton and an isoprenoid side chain and occur widely in a variety of living things, from microorganisms, such as bacteria and yeasts, to higher animals and plants. In prokaryotes, they occur in the plasma membrane and function as electron acceptors for cell membrane stabilization and for periplasmic membrane protein disulfide bond formation. In eukaryotes, they occur in the mitochondrial membrane and/or cytoplasmic membrane, and serve as essential factors in the electron transfer system in the mitochondrial respiratory chain and in the oxidative phosphorylation, function as antioxidants and contribute to the stabilization of biomembranes.
Coenzymes Q having an isoprenoid side chain resulting from condensation of 8 to 10 isoprene units, among others, have attracted attention as materials of health foods and the like. Among them, coenzyme Q10 comprising 10 isoprene units is intrinsic in humans and is therefore very useful and is in use as a heart medicine.
Commercially, this coenzyme Q10 is produced, for example, by isolating coenzymes Q from a plant, such as tobacco, and synthetically modifying the side chain length thereof.
It is also known that coenzyme Q10 is produced by a wide variety of organisms, from microorganisms, such as bacteria and yeasts, to higher animals and plants, and the method comprising cultivating a microorganism and extracting this substance from cells thereof is thought to be one of the most efficient methods of production thereof and, actually, is in use in commercial production thereof. However, such methods cannot be said to be satisfactory in productivity since, for example, the yield is poor and/or the procedure is complicated.
Attempts have also been made to isolate genes involved in biosynthesis of coenzyme Q10, amplify the genes by means of the recombinant DNA technology and utilizing them in increasing the production of coenzyme Q10. In living organisms, coenzyme Q10 is produced via a multistage complicated reaction process in which a number of enzymes are involved. The biosynthetic pathway therefor in prokaryotes differs in part from that in eukaryotes. Basically, however, the pathway comprises three main steps, namely the step of the synthesis of decaprenyl diphosphate to serve as the source of the decaprenyl side chain of coenzyme Q10, the step of the synthesis of parahydroxybenzoic acid to serve as the source of the quinone ring, and the step of the coupling of these two compounds, followed by successive substituent conversion to complete coenzyme Q10. Among the reactions involved, the reactions involved in decaprenyl diphosphate synthase, which are said to determine the rate of the whole biosynthetic reaction process and which determine the length of the side chain of coenzyme Q10, are considered to be the most important reactions.
For efficient production of coenzyme Q10, it is considered effective to isolate the decaprenyl diphosphate synthase gene, which is the key gene in the biosynthesis in question, and utilize the same for causing a production increase. Thus, so far, decaprenyl diphosphate synthase genes have been isolated from several microbial species, such as Schizosaccharomyces pombe (JP-A-09-173076) and Gluconobacter suboxydans (JP-A-10-57072) and studied for their use in coenzyme Q10 production. As for the host microorganism for this coenzyme Q10 production, it is desirable to use prokaryotes, such as Escherichia coli, from the viewpoint of productivity, safety, recombinant system preparation, and so on.
As for the decaprenyl diphosphate synthase gene sources, it is also possible to utilize eukaryotes in which coenzyme Q10 is produced in relatively large amounts. Thus, for example, fungi are strong candidates. However, when a eukaryote-derived decaprenyl diphosphate synthase gene was introduced by recombination into those microorganisms which belong to the prokaryotes, for example Escherichia coli, coenzyme Q10 was not produced or was produced only in unsatisfactory amounts. It is thought that this is due to an insufficient level of expression of the long-chain prenyl diphosphate synthase. Therefore, the development of a method of causing efficient expression, in prokaryotes, of a eukaryote-derived decaprenyl diphosphate synthase gene serving in relatively abundant coenzyme Q10 production has been desired.